Adenoviruses are linear, double-stranded, DNA viruses, from 70-90 nm diameter, nonenveloped, and having an icosahedral-shaped capside, which is formed by 240 hexons, 12 pentons and fibers extending from each icosahedral apex. These hexons, pentons and fibers represent the major adenovirus antigens, and those that determine the serotype thereof.
The adenoviruses genome is about 30-45 kb in size, and has four early regions (E1, E2, E3 and E4), and five late regions (L1-L5).
Adenoviruses have been isolated from different species, the two main genres being Aviadenovirus, isolated from birds, and Mastadenovirus, isolated from mammals.
Adenoviruses are considered good candidates as recombinant vectors for vaccine production because they are highly infectious and many of them are not pathogenic. In addition, adenovirus vectors can efficiently translate large-size genes, and generate an extended immune response in animals. Nonetheless, it is also known that the adenoviruses specific structure requires the study of the specific insertion sites of heterologous genes for each species, and that it is virtually impossible to generalize on the biological behavior of the various known adenoviruses when being converted into a recombinant viral vector.
So far, human, simian, avian, porcine, among others, adenoviruses have been utilized as vectors for potential use as recombinant vaccines. In particular, it is known that fowl adenoviruses (FadV) are potential candidates for the elaboration of recombinant vaccines. Nonetheless, as will be described below, the possibility of successfully utilizing them at a commercial level, particularly in the veterinary industry, has been generally inhibited due to the inability to reach their large-scale production with the required stability, since the production of adenoviruses is carried out from cell lines which yields are typically very poor, or which nature prevents achieving a stability of the viral vector in successive passes, whereby it is easy to understand that nowadays there are no vectorized vaccines in avian adenoviruses.
For example, International Publication No. WO94/24268 describes FAdV recombinant vectors having at least one heterologous nucleotide sequence inserted, and which are useful in generating an immune response in birds susceptible to diseases. According to this document, the non-essential regions of the adenoviral genome that may be appropriate for the heterologous gene to be replaced or inserted in, are those located at the genome right terminal end, preferably the region located between 0.0038 and 0.0039 in (97 and 99.9 m.u.) The recombinant vaccine, according to that document, could apparently be utilized in combination with vaccines against other viruses, such as that of Marek's disease or Newcastle's disease, without having evidence of this. Additionally, in this document the behavior of the adenovirus obtained after successive passes in cell lines for large-scale production is not illustrated either.
Also, U.S. Pat. No. 6,296,852 describes serotype-9 FAdV vectors (FAdV-9) wherein an insertion of nucleotide heterologous sequences into non-essential regions of the viral genome is carried out. These regions can be non-encoding regions located in the genome left and/or right end, preferably at the region located on the right end (3′), between 0.0023 and 0.0039 in (60 and 100 m.u.) As for the WO94/24268 document, although this patent identifies another wider region for the potential insertion of exogenous genes, it does not illustrate its behavior, and particularly, its integrity either, after successive passes in cell lines.
In another document (Corredor, J. C. and Nagy, E., the non-essential left end region of the fowl adenovirus 9 genome is suitable for foreign gene insertion/replacement. Virus Research. Vol 149, 167-174; 2010), it is disclosed that the 5′ end non-essential region of the FAdV-9 genome, could also be a suitable site for the insertion or replacement by exogenous genes to produce recombinant vectors. For example, one of such vectors was obtained replacing the non-essential region located between the 491-2782 nucleotides with the gene encoding for an enhanced green fluorescent protein (EGFP). This document, however, like the others, does not show whether or not the viral vectors as built would be stable when inserting an exogenous gene encoding for the antigen of a disease into them, and by replicating them in suitable cell systems to obtain a recombinant vaccine, since the document only shows a reporter gene expression and lacks in vivo assays; besides it does not mention the composition or the protection levels against a disease of interest either.
As may be seen, it is known that the non-essential regions of the FAdV genome represent potential sites where an exogenous nucleotide sequence may be inserted or replaced. However, the vectors described in the state of the art have the disadvantage of being non-stable at the time of producing recombinant vaccines at an industrial level, due to the loss of the heterologous gene inserted after various passes in cell cultures.
Further, in the state of the art, the use of adenoviral vectors in the treatment of birds, mainly poultry, has been avoided due to its interference with vaccines against Marek's disease, vaccines that are widely utilized in the avian industry. Marek's disease (EM) is a condition caused by a herpesvirus affecting domestic birds, causing a lymphoproliferative disease provoking legs or wings paralysis, and lymphoid tumors, as well as mortality. In order to prevent this disease, monovalent or polyvalent active vaccines are applied, mainly subcutaneously or in-ovo.
In order to reduce costs and achieve greater efficiency at the time of application, sometimes the co-administration of at least two vaccines is preferred. However, although documents like the above-mentioned International Publication No. WO94/24268 expose the possibility of utilizing adenoviruses in combination with Marek's vaccines; subsequent studies have demonstrated that adenovirus vaccines and Marek's virus vaccines have severe in-field interferences when mortality rate and lesions produced by both diseases on birds, preferably long-living, are analyzed, like commercial stance hens.
Currently it is now known that by simultaneously applying a whole virus vaccine against EM with a recombinant vaccine in an adenovirus vector, the vaccine against EM might interfere with the recombinant vaccine due to the following mechanisms: a) different replication kinetics; b) the vaccines compete for the same cell-type for their replication in birds; and c) the EM virus causes immunosuppression in birds. Apparently, if this interference is to be overcome, and to achieve an effective vaccination against the disease related to the exogenous gene of the recombinant vaccine, it would be necessary to administer a higher dose of said recombinant vaccine, or reduce the dose of the vaccine against EM (Breedlove et al., Avian influenza adenovirus-vectored in-ovo vaccination; target embryo tissues and combination with Marek's disease vaccine. Avian Disease. Vol. 55, 667-673; 2011). However, the same Breedlove's et al. reference shows that increasing the doses of the adenovirus recombinant vaccine could cause a temporary interference with the vaccine against EM which makes it ineffective, causing severe problems in the field. This means that, up to date, a vaccine based on a recombinant adenovirus viral vector has not been able to be formulated with an exogenous gene which does not interfere with the protection conferred by the Marek's disease vaccine or vice versa.
Likewise, maternal antibodies are another factor which can potentially cause interference with a vaccine. Although maternal antibodies confer protection to newborn animals, their presence may inhibit or reduce the vaccines effects, causing the immune-response produced thereby to be non-optimal.
Another challenge found in the development of vaccines in general is the time in which protection and duration of said protection is achieved, as a function of the formula utilized for the vaccine.
A vaccines which grants early protection, that is to say, in a very short time starting from the application, will also have a short-term effect since the levels of protection will drop rapidly, making a periodic re-vaccination necessary. In contrast, a vaccine which grants durable or long-lasting protection will take more time to achieve protection (late protection), although said protection will last for a longer time, normally requiring less re-vaccination in order to achieve the protective effect desired. Normally, to achieve lasting protection in a group of animals it is necessary to apply early protection vaccines in combination with late protection vaccines in order to avoid the possibility that the animals get sick along their entire development.
In the state of the art it is known that an active virus vaccine normally grants quick or early protection, since it achieves an acceptable, but not lasting, level of protection in a short-time range starting from its application. On the other hand, it is also known that deactivated viruses vaccines grant a more lasting protection than that of the active virus, but do not require long times to achieve it, that is to say, the protection is late and in many cases they also require re-vaccination, although less frequent.
For example, Stine et al. (Evaluation of inactivated newcastle disease, avian diseases, Vol. 24, No. 1 (January-March, 1980), pp. 99-111) assessed three vaccines against NDV: a deactivated vaccine in emulsion, a deactivated vaccine adsorbed in Al(OH)3 and an alive commercial vaccine, finding that the alive vaccine produces HI titers in chickens a week before the deactivated vaccines do, whereas the deactivated vaccine in emulsion produces a sustained immune response.
Likewise, Folitse et al. (Efficacy of combined killed-in-oil emulsion and alive newcastle disease vaccines in chickens, Avian Diseases, Vol. 42, No. 1 (January-March, 1998), pp. 173-178) mention that there are reports that the NDV deactivated vaccines, emulsified in oil, induce high and lasting levels of circulating antibodies in birds that were previously vaccinated with active vaccines. Likewise, they mention that the reason why a high antibody response is obtained upon administration of an NDV deactivated vaccine, in combination with an active vaccine against the same disease, could be due to the fact that at the beginning the alive virus replicates rapidly, eliciting a primary immune response, which is followed by a continuous but slow release of the antigen of a deactivated vaccine, which behaves like a reinforcing dose.
Also, Toro et. al (Avian influenza mucosal vaccination in chickens with replication-defective recombinant adenovirus vaccine, Avian Diseases 55:43-47, 2011) assessed the protection conferred by a recombinant adenovirus vaccine free of adenovirus competent for the replication that would express the avian influenza H5 gene optimized in its codons, which was applied on 5 days old laying type birds, and in some cases with re-vaccination when they turned 15 days old. The results showed that only the birds which were re-vaccinated developed high antibody titers. These titers could be detected starting from the age of 9 days, reaching their maximum at the age of 32 days.
In like manner, there are some active viruses that extraordinarily provide both early and lasting protection, just like the Marek virus. Nonetheless, the Marek virus can be utilized as a vector and in the state of the art it has demonstrated to achieve early and lasting protection against Marek's disease with only one application, but for the inserted exogenous antigen, it does not achieve early protection, but it only presents late and lasting protection.
As can be seen from the state of the art, it has not been possible to successfully utilize adenoviruses and, in particular, serotype 9 avian adenovirus FAdV-9 in the state of the art as a recombinant vaccine for birds with any exogenous gene at an industrial scale, although diverse insertion sites have been described, on the one hand due to the fact that adequate stability is not achieved in their reproduction on cell lines after successive passes, and on the other hand due to the fact that the Marek's disease vaccine interferes with the action mechanism of adenoviruses or vice versa, reason for which the use of adenoviruses in combination with Marek's disease vaccines has been expressly avoided in the state of the art. Further, it has not been possible to obtain a viral recombinant vaccine which achieves both early and lasting protection for an exogenous antigen inserted in the viral vector utilized with only one application.